Abstract Introduction: We have developed an assay that distinguishes CA125 originating from malignant and benign sources. The assay combines conventional immunoassay with the detection of carbohydrates differentially glycosylated in epithelial ovarian cancer (EOC). These glycans can be detected using carbohydrate-reactive lectins. Our recent data showed that CA125 detection using MGL-lectin improves the discrimination of cancerous CA125 from CA125 of endometriosis patients (Gidwani et al., Clin.Chem 2016). The current study extends CA125MGL measurements to a broader sample set and longitudinal collections during EOC treatment and follow-up. Methods: MGL lectin coated onto fluorescent europium-chelate-doped 97-nm nanoparticles was used for detection of the CA125 that was captured on an anti-CA125 antibody immobilized on microtitration wells. Serum samples from high-grade serous EOC or patients with varying benign conditions and healthy controls were analyzed. Results: CA125MGL specifically recognized the CA125 isoform produced by EOC, whereas the recognition of CA125 from nonmalignant conditions was reduced. Serum CA125MGL measurement discriminated better patients with EOC from benign conditions compared with conventional immunoassay. Importantly, discrimination was particularly improved for marginally increased CA125 values that are common in benign conditions. Our data also showed earlier detection of EOC progression, suggesting that the CA125MGL also has potential to improve earlier diagnosis of EOC. Conclusions: The CA125MGL concept could help reduce the false-positive rates of conventional CA125 immunoassays. It carries further potential to improve early diagnosis of EOC as well as follow-up of treatment response. Finally, using appropriate combinations of lectins and antibodies, applying the test concept presented here can also be explored for other diagnostic targets, where changes in glycosylation are indicative of an ongoing disease process. Citation Format: Kaisa Huhtinen, Kamlesh Gidwani, Henna Kekki, Johanna Hynninen, Liina Salminen, Antti Perheentupa, Matti Poutanen, Annika Auranen, Seija Grenman, Urpo Lamminmäki, Olli Carpén, Kim Pettersson. A cancer-specific detection of serum CA125 improves differential diagnosis of epithelial ovarian cancer from benign conditions. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B15.